Metabolic Reprogramming of Iron Endows the Function of ISCA2-TET Interaction During Peri-implantation Development (EM-Seq)

Metabolic reprogramming plays important roles in embryo development. However, the metabolic network has still not been systematically investigated during the processes. In this study, we develop MFE, a metabolites flux estimation tool, to predict metabolite abundances and their regulons, highlighting the role of Fe(II) and its regulon-ISCA2, in the inner cell mass (ICM) of human and mouse blastocysts. By introducing Isca2-/-, Isca2G77S/G77S and Isca2C144S/C144S mice, we find ISCA2 binding with iron, but not Fe-S cluster, is indispensable for mouse peri-implantation development. Mechanistically, nuclear ISCA2 interacts with TET, and Fe(II) is important for the interaction by triggering the enzymatic activity of TET. The partnership safeguards promoters of epiblast-specific genes from hypermethylation and is essential for epiblast development in mouse peri-implantation embryos. The study provides insights into how iron metabolism modulates embryo development, and suggests a manner of Fe(II)-dependentenzymes to achieve activity under physiological conditions. Overall design: RNA-seq, small RNA-seq, EM-seq of mouse Isca2 WT and KO pre-implantation embryos. To collected pre-implantation embryos, Isca2+/- female mouse intraperitoneal with 6.5 international unit of pregnant mares' serum gonadotropin and, 45 to 47 h later, with 5 IU of human chorionic gonadotropin and crossed to Isca2+/- males.代谢重编程在胚胎发育中发挥关键作用，但目前该过程中的代谢网络仍未得到系统研究。本研究开发了代谢物通量估测工具MFE（metabolites flux estimation tool），用于预测代谢物丰度及其对应的调节子，重点揭示了Fe(II)及其调节子ISCA2在人类和小鼠囊胚内细胞团（inner cell mass, ICM）中的作用。通过构建Isca2基因敲除（Isca2-/-）、Isca2G77S/G77S及Isca2C144S/C144S突变小鼠，本研究发现，ISCA2与铁离子结合（而非结合铁硫簇）的能力，对小鼠着床期发育不可或缺。机制研究表明，核定位的ISCA2可与TET蛋白相互作用，而Fe(II)可通过激活TET的酶活性调控这一相互作用。二者的相互作用可维持上胚层特异性基因启动子区域免于过度甲基化，对小鼠着床期胚胎的上胚层发育至关重要。本研究揭示了铁代谢调控胚胎发育的潜在机制，并为Fe(II)依赖型酶在生理条件下如何发挥活性提供了新的理论视角。实验整体设计：对小鼠Isca2野生型（wild type, WT）及基因敲除（knock out, KO）型着床前胚胎进行RNA测序（RNA-seq）、小RNA测序（small RNA-seq）及EM-seq检测。为收集着床前胚胎，研究人员对Isca2+/-雌性小鼠腹腔注射6.5国际单位的孕马血清促性腺激素，45至47小时后再注射5 IU人绒毛膜促性腺激素，并与Isca2+/-雄性小鼠合笼交配。